X-FISH: Analysis of cellular RNA expression patterns using flow cytometry.

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Fluorescent in situ hybridization (FISH) links molecular and cell biology but faces challenges in flow-based platforms.
• Existing FISH adaptations are limited by procedural complexity and high costs of commercial kits.

Purpose of the Study:

• To present a modified protocol for sensitive and specific RNA detection in cells using flow cytometry (expression-FISH; X-FISH).
• To demonstrate X-FISH's utility in monitoring mRNA expression changes, combining RNA and protein analysis, and distinguishing cell subsets.

Main Methods:

• Development and application of a modified fluorescent in situ hybridization protocol for flow cytometry (X-FISH).
• Integration of X-FISH with antibody staining for multiparametric analysis of RNA and protein.
• Application in primary cells and cell lines to study mRNA expression dynamics and cell population heterogeneity.

Main Results:

• X-FISH enables sensitive and specific detection of RNA expression patterns in various cell types.
• The technique successfully monitors mRNA expression changes upon activation and distinguishes cell subsets.
• Combined RNA and protein analysis is achievable within the same sample, enhancing multiparametric studies.

Conclusions:

• Expression-FISH (X-FISH) offers an economical and efficient method for RNA analysis via flow cytometry.
• X-FISH provides a valuable alternative to antibody-based markers, especially for non-classical animal models.
• This technique enhances statistical robustness and applicability across conventional and multi-spectral imaging flow cytometry platforms.